I have collected synthetic proteins from E.Coli. Then run SDS-PAGE. The problem is when calculating insilico its size should be around 64kDa. However, when running on the ladder, it is below the 60kDa line of the ladder.
Has anyone experienced this phenomenon and have a way to explain it?
I double checked through sequencing the expression plasmids in e.coli and they are absolutely correct.
It is not so uncommon to have proteins running anomalously in SDS-PAGE. Bands run according to their mass and net charge, the latter provided mainly by SDS. If your protein is acidic (low isoelectric point, pI), this will increase its migration due to the greater negative net charge in comparison to the ladder. That could be the explanation.
Totally normal. Even the small Sumo protein migrates differently on SDS-PAGE. If you are unsure, you could submit your sample for intact mass spectrometry to get the exact mass of your purified protein. I recognize mass spectrometers are kind of expensive but if your facility has one, it is accurate to plus or minus like 3 protons.
This could be due to SUMOylation. SUMO (Small Ubiquitin-like Modifier) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. I recommend you check its function. Usually, SUMO proteins are small; most are around 100 amino acids in length and 12 kDa in mass.
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